Je sept Mot of te parmactn mpmund a © gt ets a a Sarr Seay sy a foe corr or cs oe se enema cyan ees ae ee ee a tn poms meen ite ae ae ee St ere as lochemieal seperations lc isolation of some drugs or metabolites {fom blood, urine etc. concentration of lonle solutions: A cation or anion from a balk Stroluon ean be adsorbed onto fon exchange resin. After adsorption, ft can be eluted ty using small volume of eluent. 8, ton exchange columa in HPLC: For separation of compounds of ‘mixed nature lke acile and basic substances, fon exchange eslumn, {i used in HPLC (High performance lguid chromatograpty). -_ sew 1 Prine of Separate ' Criteria or analas by GC - Volaully and Thermestabaty ‘© Practical Requirements 9 Carmer gas - Ha He, Me Flow regulators and flow meters Rotameter & Soap bubble meter Injection devices (Columns ‘Analytical and Preparative pe Packed, Open tubular, SCOT columns ‘Temperature contre devices Twothermal and Linear programming Detectors ‘Thermal Conducuoty detector Flame loreation detector ‘Argon Tonsstion detector Eeeton Capture detector Recorders and ineyato © Dervatisation of sample - Preohumn & Post column © Pretreatment of slid support ‘Parameters used in OC “Retention ume, Retenton volume. Separation factor Resolution, Theoreieal plate, Bictency Asymmetry factor © Applications of Gas Chromatography eaINTRODUCTION eee oo SE ESS eas oe eS, ily pa ‘Osc 1s mot widely used beense of lmted number of stato, pias Slate: 8G te prinile of separation is adsorption. GS pres ere irre thre a leas sll of shun it stations 4 ea nrc Therefore all te lacuaslona thls chapter rey, to @Uc technique only PRINCIPLE OF SEPARATION ‘The principle of separation in GLC 1s partition. Gas Is used as mobile phase. Liquid which Is coated on to.a solid support is used as ationary phase The mixture of components to be separated 1s converted ‘o vapour and mixed with gaseous moblle phase. The component which 4s more soluble in the staonary phase travels slower and eluted later, ‘The component which fs lees soluble In the stationary phase, eaves faster fand elute out fst. No two components has the same partion co-eicent far a fed combinaten of staionary phase, mobile’ phase and other cindiuons. Hence the components are. separated according t0 ther Darttion coefficients, (Partition co-efficient is Une ratio of sclubihty of Seubstance isirbuted between two tmmiscble liquids at ® constant Texperature) CRITERIA FOR COMPOUNDS TO RE ANALYSED BY ‘Two important eteia are 1, Volatility: Unless a compound is volatile, it cannot_be mixed with ‘mobile phase, Hence volatility is important. 2 Theemostabilty: All the compounds will not be in the form of vapour. There wil be solid as well ar liquid samples, Hence (0 convert them fo a vapour form, Uhey have to be heated to higher ‘emperature. At that temperature, the compounds have to be thermostable. Wf they are not thermostable, the compounds cannot te analysed by Gas chromatography, ince they wil be decompose PRACTICAL REQUIREMENTS carer gas Flow regulators and tow meters Injection devlees ‘Cams Temperature cont dors Recorders end Integrators Goes Chromatographie Apparstas ara SS “| 2. CARRIER GAS, “The choice of carer gat determines the eltceney of chromatograple separation. Most widely used caver gases are Hydrgen, Helium, Nitrogen ‘and Argon ‘Hydrogen: It has better thermal conduct, ow density. 1 use In case of thermal conductnty detector and fam tonisation detector. The ‘isadvantage ts that it reaca wih uneturate_ compounds and it ts {inflammable etiam: 1 also has excelent thermal conductivy, but i is expensive, tee a good camer ans when used with term conductvty detector Nitrogen: Its inexpensive but has reduced sensitivity. 173Requirements of a carer gas: 1. inertness, 2. Suitable to aye ‘detector used, 8. High purity. 4 Easy avalable, 5. Cheap, 6. Less et of explosion or fre hazards, 7. Should give best column performanst consistent with the required speed of analysis, Considering these requirements, and a compromise among. inertness, ficiency and operaing cos, etc make Nitrogen and Helium a8 the mon formen earner £25 ‘As carler gas Is compressible, gases are stored under high pressure fn finders and used when required, 2, FLOW REGULATORS AND FLOW METERS ‘As carver gases are stored under high pressure, flow regulators are used to deliver the gas with uniform pressure or ow rate. Flow meters are used to measure the fow rate of carter gas. They are Rotameter and Soap bubble flow meter. ‘Rotameter: It is placed conveniently before the column inlet. It has fan ordinary glass tube (uke burete) with a float held on 0 a spring. The level of the float 1s determined by the flow rate of carer gas and ts recalibrate — Seap bubble meter: it is simar to rotameter and instead of ‘afloat, soap bubble formed indicates Whe flow rate, Tt has a. glass tube with @ inlet tube at the otto ugh which gas. comes In. A rubber bulb is used to store soap solution. When the bulb le. gently rested, a drop of soap solution ie fcomerted into’ a bubble by the Dressure of earier gas and travels Ube une ‘up. The distance travelled upwards "¢7'2 Mme la measure of fow rate of carer fas. The graduations are "also precalbrated, m/f 14 ‘3. BUECTION Devices Samples for introducing ino the cota caver a2, UQUld OF sold nator Gases can be Anoduced into the calumn by vabe devices can be of any ype Le, Solid samples are dissolved in a suitable solvent an = eae able solvent and then they 4, COLUMNS CCohumn is one ofthe important part of GC which decides the separation efiieney, Columns are made up of ats or stainless see Stanioes ste fofumns have the advantage of log lle and ean be easy handled without the fear of fragility. But some samples reat with ter, Hence in such eases, glass columns are used. ef, Strolis, Glass columns have the advantage that they are Inert and do not react wth the any ind of Sample. The great disadvantage Ye that they are highly Sagle and. are (illeul to handle ‘Columns can be clasited according to the nature as well as ts use, ‘A. Depending on its se 4 Analytical column: Anabyical columns have a length of 1-15 metres and an outer diameter of 8-6 mm. They are packed columns ‘and are made up of glass or stainless ste. Oniy small quantity of samples can be loaded on to the lum 1s, Preparative columa: Preparative columns are larger when compared to anata) columns since lange amount of sample has to be loaded. ‘They have a lengih of 8-6 metres and outside dlameter of 6- Orn, 15B. Depending on ite mature 4. Packed eolamm: Columns are avalable in packed manner Commercially and hence are called as packed columns. Ditlereng Cclumns ranging from low polar nature to high polar nature ae (evalable, Examples of such columns, operating temperature ‘ppliealons of such columns are given in the following table Statlonary phases for GLC: A wide varity of stationary phases te Patyetiylene gens, high molecular weight esters, amides, lydrocarbong, palystlatanes, microporous crosslinked polyaromatic beads. ‘satenny pe ate aa [vane wore Now polar 40" to 826% [Pubicphenydmetiy sloane| Now-polr bended pase | 60" to 800hc Porano propyl phen! Intermediate poly ‘upto 250° fare store Favanvine eyes! Re 3 to Bae [svete evel Poke 50" we 260°C 0 moaned wats Tor bonded phase | 60" to 200°C IMeroarepaae acd [Poy te xno prop ana) Very polar Nowbanded phase | upto 250° | 4 Open tabular column or capilary column or Golay column: Thcy are made up of long capllay thing of 80°90 metres in length land hve uniform and narrow internal dameter of 0.025 0.075 em. ‘These are made up of stainless steel and are tn the form of 4 cl ‘The inner wall of the capllay 1s coated with the stationary phase guid in the form of a thin Mm (05 to IW). Theor columns. oer least resistance to the Dow of carrer gas and hence they are more fefent than packed columns which ‘offer more reslsiance to the ‘ow of carrier gas. But the disadvantage that more sample cannot be leaded. ‘ML SCOT columns (Support Coated Open Tubular Columa): This 1 fan improved version of olay or eapilary columns. As olay. or capillary columns have small sample eapacty, they can be mosifed Into SCOT columns 16 5, TEMPERATURE CONTROL Devices Preheaters: Prchesters are used im Gas chromatography to convert the sample into its vapour form and mix them with tie reote phe feamier gas. The preheaters are present along. with Injecting, devices, Ac son as liquid samples are injected, they are convert inte nares bee ‘Thermostatieally controlled oven: The principle of separation in Gas _twomatography is partion. Partition eo-feient is the rao of coneenoaten of solute datibuted between two immiseibie luda, Since portitiog ot ‘Gfelent a5 weil as solubility of a solute depends upon temperarun, temperature maintenance in « column t highly essential fr ciney, separation, Hence the column as well as injerting dovees onan ee ‘atnained at a particular temperature ‘As columns are long. they cannot be enclosed in oven easy. Hence the columns are in a cotled form and enclosed in thermostaclly tent ohed oven. ‘These ovens are highly accurate and can mainate: teapots pearest 10'0.1°C. ‘Two types of operations are avaiable. They are | Wothermal programming: (iso means same) in which the same temperature is maintained throughout the process of separation, 4 tnear which the oven Is heated Hinearly over a period of time. eg. 150°C initially t 200°C at the end of separation with an increase’ in temperature at the rate of 5°C/minute, Ths ‘ype of near programaung Is required when a sample has tbsure of low bolling and high bollng’ point compounds, This stethed Is fffclent for separation of such complex mixtures Detectors are the mest important part of gas chromatographic ‘struments, They are considered as heart of the apparatus, A detector‘uses some property by which st can detect the diference between & pure fearrer gas and a eluted component ‘Toe requirements of an ideal detector are: 1. Applicability to wide range of samples. 1s High Senstuvigy to even small concentrations ML Rapiity of response, ve Uneasy: Le, less response to low concentration and propertonal ‘reaponse to high concentrate. ‘% Responst. should be unaffected by temperature, flow rate or characterises of carrer EAS. Non destrucive tthe sample in case of preparative work: ‘Simple and easy to maintain vil Inexpensive. ‘The diferent detectors wsed commonly are ‘a Katharometer or Thermal Conductivity Detector (10D) ©, Flame foisaton Detector (FD) © Argon lonlaslon Detector (AID) 4. Electron Capture Detector (ECD) ‘. Matharometer or Thermal conductivity detector ‘The principle 15. based upon thermal conductivity °F 2 owe ‘ference between carver gas fand. that of component. (Ig. men 17-4, Kathacometer has t¥o platinum sites of uniform mensions which form part ‘of Wheatstone bridge. Through fone of them, pure carier gas "sre a fiways ows through and = Sse Ahrough the other, the eluents of the column passes. The two platinum wires are heated electrically and fence assume equlbrium condiuons of temperature and electrialrelstance (when pure carer gas passes through both of them. there 1s no difference I temperature or resistance and hence a bastine is recorded. When a fompanent emerges ffom the column, it alters the thermal conductvly fod resistance of the wire. Hence this produces a diference in resistance nd se conductivity between two wes, which 1s amplifed and recorded ea signal ‘The thermal conductviles of some easier gases are gen as follows: a_| He | Nz | Methane | Hesne m7 [sol sa] 6s | 30 ‘From the tabular column, i ean be seen that Hydrogen and Hettum have higher thermal conductivigy and they are the best carier gases for Kaharometer Hydrogen is inflammable end helum is expensive. But both Of them sffer good response. Kany other carer gas is used. they ve fise to neghive peaks Because of lower thermal conductivity. Advantages of Katharometer Applicable to mest compounds 1 Linearity 9 ged, {The sample not destroyed & ence used in preparative scale 1, Simple easy to matntain and inexpensive Disadvantages of Katherometer 1 Law sens. 1 Affected by Quctuntions in temperature and flow rte 1u. The response is ony relative and not absolute ‘Biological samples cannot be analysed1 Mame enlaton detector (FD) = ‘te joastlon eect -_ vased won ne esr eoecty ‘ss a etn gu mt ‘Spoutre bat Doxowe ond Time a pen corer ga sed wih ape srt noe gen Tt Ue caer pris ler rirogen ot yon can te ied ws hyrogen nrreeh the burner up-made up ‘patna caplay, which acts ree fe gece (aod, The anode ng. remnant OO fester gauze placed tle above Tamer up. When pure carrer gos alone pases, there Is no tonisation Sod no current fows. When @ component emerges trom the column, ‘umber of ona are produced because of tonsation by the thermal enrey the Gane This causes a plea difrence and causes a Now of urret which (9 amplified and recorded a8 signal (Fig 175) Advantages 1, This detector Is extremely semaine and background noise $6 low. ence wg quaniiles of the solute can be detected 2. Sable and insenstive to small changes tn the ow rate of carier (i and water vapour, 8, Responds to moet of the organte compounds 4 Meany Ie exelent, ©, Argon Jonlaatlon detector (AID) ‘Tis pe of detector depends on the extiaton of argon atoms to @ rmctastble sate, by using raoactve energy Tis le chloe by evading ‘he corner eas with ltber a parties of particles, « particles can be btaned form radhum-D. P parties cane obtained from ® Sy or i 1710 ‘These high energy parties fonise the argon atoms and hence they sue exited to metastable stale, These molecules collide with the effluent ‘Golecles and ‘onises them, These lons when they reach the detector will use aa increase In current. Thus the compounds can be detected, ‘The serles of events which take place in this detector Is given as ‘slows pe A gm + SSE, ee te avenge 1 Rerpnds to mot ofthe ore compounds 2 Sem ey ie Disadvantages 1. Response ts not absclute and 1 is relative. 2. Leary 1s poor Sensitivity 1s affected by water and is much reduced for halogenated ‘compounds. ‘The response varies with the temperature of the detector and for high temperatures Ike 240°C, vltages of 1000V or less are usually necessary. 4, Hlectron Capture Detector (ECD) ‘The electron capture detector (Fig 17.6) has two electrodes, with the column fluent passing between them, One of the electrode ts eated = ‘wi a radioactive Isotope which emits electrons 88 1 decays, These emited electrons produce secondary electrons which are collected by the ‘ode, wien potential of 20V te applied between them.'When carrier gas slone flows through, all the secondary electrons are collected by te weanIhe ponthely polarised electrode. Hence a steady baseline is recorded, [Egfiuent molecules which have ality fr electrons, eqpture these electrons ‘Shen ey pass tough the electrodes. Hence the amount of steady state Thurent i reduced. This diference 1s amplified and recorded a3 output signal “The carr gas used in Uns typeof detector depends upon the electron aftity of the compounds analysed. For compounds with high electron Stig, argon is used as oarer gas. For compounds of low electron ‘iy, altogen. hydrogen, helium or carbon dioxide can be used as caer Eas ‘The advantage of this type of detector 1s that st 18 highly sensitive. ‘Bren nanogram quantiles can be detected, But the disadvantage te that {can be used only for compounds with electron ality. Halogenated ‘compounds, several pesca, etc can be detected using this (pe of ‘detector. ‘Compartzon of the Sensltivity of Detectors ee ] —- ocae 7, Recorders and Integrators [Recorders are used to record the responses obtained from detectors ‘ater amplification, necessary. They record the baseline and all the peal Stained. with respect to Ume. Retention time forall the. peaks can be ound out from sues recordings, but the area of In ann ws tidal peaks cannot Integrators: Integrators are improved version of recorders with some data provessing capabilities. They can record the individual. peaks wit retention time, aight and width of peaks, pele area, percentage of area, fe, Integrators provide more information on peaks that recorders, waa [DERIVATISATION OF SAMPLE Dertvaisaion is a technique of treatment of the sample to improve the process of separation by column or detection by detector. There are two pes based upon tts need. They are Precoluma dertvatistion: Ths 1 dane to improve some properties of te semple for separauon by column, By this dervausation technique, tbe components are converted to more volale and thermostable dertvaives. Narcover improved separation and less taling wil be seen aller such treatment In the following conditions, pre column dervatisation ts done. 1. The component is less velo 2. The compounds are thermolabil, Le, heat sensitive 1 To reduce taling 4. To improve separation facto. Example: Carboryile acids, sugars, phenols, alccls, ete can be ‘Conered to Tess polar compounds by using reagents like BSA reagent {Bis trimethyl Syl Acetamide reagent. ‘they can also be convered to acetyl dervaive or tilouro actyt ert. Post column destvatisation: Post column dervalisation t= done to Improve the response, ahown by detector. The components may not be Teeted by detector unless dertalsation 1s done The components may be converted im such away that ther sonisation or ait towards electrons fe increased, Normaly this is "OnLine detection technique where the Aowrate te nelther stopped nor altered PRETREATMENT OF SOLID SUPPORT ‘Solid support is used to hold the stationary phase tiqud as a thin fm, Dut someumes due to some defects, uniformity and stably of the flim of ligad satonary phase may net exist. In auch eases talling of Peaks anew separation efficiency can be observed. Therefore to overome Such demerits, itis best {odo pretreatment of the stalonary phase. was _—ent a ing pens er wpe ee ce i ng un CaS eae 1. By slag more polar quid stationary phase 12, Increasing the amount af quid phase on the support. 3. By selecting a ess active support. 44 Preveaument of the slld support 10 remove active sites, ‘Bxample: By using hecamethyl dsllazone or dimethyl sly! dichiorde ‘This converts te hydroxy groups (polar) to dimethyl group (non Palas) and hence the acdve adsorption sites are deactivated Het OS Retention time (2) Retention time ie the diference in time between the point af injection and appearance of peak maxima, Retention me Is the ume required for 50% of a component Famtot to be duted fom a estomn, Retention time Seen ‘is measured in mines or seconds. Retention ge Sk time Is also" propordonal to the stance ‘moved ona chart paper, which can be Teaston Gat ‘measured nom or cn. ‘Retention volume (¥) Teenton vue the volume of ete gas requed to ete 50% ot the component fea the ca, We the predse of recta fme Retenlon volume = Retenton ume » flow rate separation factor (8) Separation factor la the ratio of patton co-ficlent of the two components to be separated. It can be exprested and determined by using the following equation: po eere ee KR tty where ty = Retention time of unetained substance iy Ka = Partition coefcients of b and a ‘oe te = Retention ime of substance b and a $= depends on liquid phase & cokumn temperature there le more diference in partition coefclent between two compounds, the peaks are far apart and the separation factor is more. If the partition neficients of two compounds azesimiar, then the peaks are closer and the separation factor i less Resolution ie & measure of the extent of separation of two components nd the baseline separation achieved. It can be determined by using the flowing formal: 2(Re-Ri a 17s— ee, ‘Teeoretical Pate (Plate theory) ‘A theoretical pate is an imaginary or hypothetical unit of & coksmn ‘shee ditbuton of solute between stationary phase and motile phase ho tained equlibrium, A theoretical plate can lao be called as functional suk of he oka, [METP -telght Equivalent to 4 Theoretical Plate ‘A theoretical plate canbe ony height, which decides the eeiency of cepratio. If HETP is tes, the column ie more efiient. I HETP ie more, the cohimn i less ecient ETP can be calculated by using the following forme: even eth ct ETP pen yh Vas Det eine where A= Edy aifuson term or muitipe path diision Which aries doe to pecking f the column, The ‘i unafoced by carter gua velocity o fw eat ‘his can be minimised by undrmiyIn pacing. B= Longitudinal ditusion term or molecule dision ‘which depends on fow rate © Bet of mass wansfer which depends on fw rte 1 Plow rate or vl ofthe mobile phase ‘Te following gure s the elect of Now ‘ait on HETP. A column Us @felent only Shot HETP ts minimum. Hence an ideal rate corresponding to the minimum {alue of HET is used pitctency (No. of theoretical plates) ciency of a column to expressed by te mumber of Ueoretial plates. I can be Sermimed by sing the formula Re n= 685 no. of Uheoretieal plates Deak wth at base i and w are measured in common units rm or em of minutes or seconds) and are proportional to the distances marked on chart paper the mumber of theoretical plates Is high, the column is ssid to be Righly efficient. If the numer of theoredal plates ts low, the ealumn 1S sald 9 be less eficient. For gas chromatographic columns, a value of {00/metre ie efctent. But in HPLC, hgh values ike 40,000 to 70,000/metre se recommend, ‘Armmetry factor ‘A huomatograple peak should be eymmetscal about is centre and ‘aid to fellow Gaiesian disuibuton. In such eases, the pele wil be like fm loc tangle But In peat, due to some factors the pene 0 aymetiesl and shows tling or fronting as shown tn the following Seen, Pronting is due to saturation of stallonary phase and can be avoided "y using ese quantity of sample. Taiing is due to more acure adsorption«Nath 2A Se ame ve sites and can be eliminated by suppor pretrentment, more polar me sed inreasing the amount of gud phase. ‘Asymmetry factor (0.95 10 1.08) can be calculated by using the forme, AF = b/a fo and 8 calculated at 5% or 10% of the peal Heit) [APPLICATIONS OF GAS CHROMATOGRAPHY 1. Qualitative analysis: It nothing but Weniieation of @ compound seat coy comparing the retention time of the sample as Wal ar the standard: Under iene conditions the retention ime W the standard and the sample are same. Wf there ts a deviation then tiey are not the same compeund. 12 Checking the puity of «compound: By comparing the chromatogram Stine standard and that of Use sample, the purty ofthe compound (Gan be reported, Wf addldonal peaks are obiained, impures are resent and hence the compound fe not pure. From the percentage rea of the peaks oblained, the percentage purtty can also be reported, 2, Presence of impariles: This can be seen by the presence of ‘ditional peak when compared wilh a standard o reference materi ‘The percentage of opuren may also be calculated from peak areas “4 Quenltatire anya: The quant ofa component can be determined By soveral methods like ‘2 Dicect comparison method By injecting a sample and standard separiely and comparing ther peak areas, the quan of the sample can be determined. ‘Area of the peak = peak eight x wath of peak at the half het where Ai and Az are peak area of sample and standard Wh and Wa are welght or concentration of sample ‘and standard is the response factor , Calibration curve method 1m ealloration curve method, standard of varying concentrations are used to determine thelr peak ares. A graph of peak area Vo concentration tthe drug te plated. From the peak arca of the unknown sample, by Iitvaplaion. the concentration of the sample can be dlermined. This fnetiod has the advantage at eos, i any are minimised ©, Internal standard method mn Chis method, @ compound with similar retention characteristics is ‘used. A known concentra of the tnteral standard is added separately to the standard solution and sample solution whose concentration ts not ‘knawn. The ehtomalogram la recréed and te peak area rao of standard land internal. standar@ is. determined. By using the peak area ratlo of Stmple and intemal standard, the concentration of the unknown soluon {determined This method i sell when more extraction steps are lnvelved in sample preparation and the sample mat 16 complex. 5. Multicomponent anaists or Determination of minture of drugs ‘Sina to the quantifestion of singe dug. mulcomponent analysis fan also be doe exay, The quantity af each component is determined ty using any one of the above methods. Marketed formulations are trailable wich contain several drugr and each component can Be Getermined quantitatively 6, Holalon and Mention of drugs or metabolites in urine, plasms, ferum ste can be cared Ov 7, twolaon and identieation of mixture of components Uke amino cis, plant extracts, wale ol, tc wagSome of the pharmaceutical appiteaions are described below: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) 1. Punty of compounds Dee ‘Column [Temperature | internal Standard | © introduction han mht 220° onatropine = Comparison of lasscal column chromatography with HPLC Fete fostwns 2006] Hottie © Types of HPLC techniques lane Ikon a0 A. Based on modes of chromatography scapotmine —[Phenimetot lana Normal Phase and Reverse Phase mode [focebrmde _[seone © B. Based on Principle of Separation ieee aerate el eel omens ‘Adsorption, lon exchange. Ion patring, Size exclusion Dees Purpose | Column [Temp] internal standard or Gel permeation. lity and Chiral Phase [Syiosrend|i7asinetran-\7} 20 11 -